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Phytomedicine
journal homepage: www.elsevier.de/phymed

Ginsenoside Rg1 provides neuroprotection against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of aquaporin 4 expression
Yun Zhou 1 , Hui-qin Li 1 , Lin Lu 1 , Deng-lei Fu, Ai-ju Liu, Ji-huang Li, Guo-qing Zheng
The Center of Neurology and Rehabilitation, The Second Afliated Hospital of Wenzhou Medical University, Wenzhou 325027, China

a r t i c l e

i n f o

a b s t r a c t
Ginsenoside Rg1 is regarded as one of main bioactive compounds responsible for pharmaceutical actions of ginseng with little toxicity and has been shown to have possibly neuroprotective effects. However, the mechanism of its neuroprotection for acute ischemic stroke is still elusive. The purpose of present study is thus to assess the neuroprotective effects of the ginsenoside Rg1 against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion, and then to explore the mechanisms for these neuroprotective effects by targeting aquaporin 4. Focal cerebral ischemia was induced by middle cerebral artery occlusion. Neurological examinations were performed by using Longas 5-point scale. Evans blue dye was used to investigate the effects of ginsenoside Rg1 on blood brain barrier permeability. Immunohistochemical analysis and real-time uorescence quantitative polymerase chain reaction were used to assess aquaporin 4 expression. As a result, general linear model with repeated measures analysis of variance for neurological scores at 5 repeated measures showed that ginsenoside Rg1-treated group could signicantly reduce the changing trend of neurological decit scores when compared with the middle cerebral artery occlusion model group (p < 0.05). Compared with the middle cerebral artery occlusion model group, ginsenoside Rg1 group has signicantly decreased Evans blue content and reduced aquaporin 4 expression at each time point (p < 0.05). In conclusion, ginsenoside Rg1 as a ginsenoside neuroprotective agent could improve neurological injury, attenuate blood brain barrier disruption and downregulate aquaporin 4 expression induced by cerebral ischemia/reperfusion insults in rats. 2013 Elsevier GmbH. All rights reserved.

Article history: Received 26 August 2013 Received in revised form 24 October 2013 Accepted 20 December 2013 Keywords: Stroke Ginsenoside Rg1 Neuroprotection Blood brain barrier disruption Aquaporin 4

Introduction Despite stroke declined from the third to the fourth leading cause of death in the United States after heart disease, cancer, and chronic lower respiratory diseases in 2008 (Towghi and Saver 2011), stroke is still one of leading causes of death worldwide and the rst cause of acquired disability, and its costs both direct and indirect are astronomic (Strong et al. 2007). Ischemic stroke refers to focal brain infarction that produces sudden neurologic decits persisting for longer than 1 h and accounts for

Abbreviations: ANOVA, analysis-of-variance; AQP4, aquaporin-4; BBB, bloodbrain barrier; EB, Evans blue; MCAO, middle cerebral artery occlusion; OD, optical density; PCR, polymerase chain reaction; rt-PA, recombinant tissue plasminogen activator. Corresponding author. Tel.: +86 13566288727; fax: +86 577 88832693. E-mail address: gq zheng@sohu.com (G.-q. Zheng). 1 These authors contributed equally to this work. 0944-7113/$ see front matter 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.12.005

8085% of all strokes (Zheng et al. 2010). However, up to now treatment has been largely restricted to supportive and rehabilitative care except intravenous administration of recombinant tissue plasminogen activator (rt-PA). Intravenous rt-PA remains the only Food and Drug Administration approved pharmacological therapy for treatment of patients with acute ischemic stroke, but intravenous rt-PA is recommended for selected patients only up to 4.5 h after stroke onset (Jauch et al. 2013). Furthermore, symptomatic intracranial hemorrhage is a devastating complication of intravenous thrombolysis treatment that is associated with high mortality (Seet and Rabinstein 2012). Another specialized care for stroke is neuroprotective agents. Neuroprotection refers to the concept of applying a therapy that directly affects the brain tissue to salvage or delay the infarction of the still-viable ischemic penumbra, rather than reperfusing the tissue (Jauch et al. 2013). Thus, neuroprotection could reduce the ischemic impairment and the overall costs that are accompanied in patients with severe disability. Pharmacological neuroprotection agents that limit the cellular effects of acute ischemia or reperfusion may limit neurological

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(Papadopoulos et al. 2004). However, the role of AQP4 in ischemic edema is still in contrary. In the present study, we assessed the neuroprotective effects of the Rg1 against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion. Furthermore, the mechanisms for these neuroprotective effects were explored by targeting AQP4.
Fig. 1. Chemical structure of ginsenoside Rg1.

Materials and methods injury after ischemic stroke (Jauch et al. 2013). There has been a recent explosion of interest in neuroprotection for ischemic stroke, with over 1000 experimental papers and over 400 clinical articles appearing from 2001 to 2007 (Ginsberg 2008), but at present no pharmacological agents with putative neuroprotective actions have demonstrated efcacy in improving outcomes after ischemic stroke (Jauch et al. 2013). Therefore, many researchers resort to naturally occurring compound as an potentially neuroprotective agent. Radix Ginseng, the root and rhizome of Panax ginseng C. A. Meyer, has been used as a representative tonic remedy in China and elsewhere for over 2000 years (Nah et al. 2007), and now is still one of the most commonly used healing herbs for stroke and chronic debilitating conditions (Zheng et al. 2011). The main pharmacologically active ingredients of ginseng are ginsenosides, being responsible for most of the activities of ginseng (Lu et al. 2009). Over 30 ginsenosides have been identied and classied into two categories as follows: (1) the 20(S)-protopanaxadiol (PPD-type: Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, Rs1) and (2) the 20(S)-protopanaxatriol (PPT-type: Re, Rf, Rg1, Rg2, Rh1) (Leung and Wong 2010). Ginsenoside Rg1, which chemically belongs to the PPT ginsenoside group (Fig. 1), is regarded as one of main bioactive compounds responsible for pharmaceutical actions of ginseng with little toxicity and has been shown to have possibly neuroprotective effects (Jiang et al. 2012). In recent years, many benecial effects of Rg1 on the nervous and vascular systems have been reported as a potentially neuroprotective agent (Nah et al. 2007). However, the mechanisms of its neuroprotective effects for ischemic stroke are still elusive. The bloodbrain barrier (BBB) is an important regulator of brain homeostasis, and its disturbance has been implicated in the onset and/or evolution of many pathological manifestations of stroke (Borlongan et al. 2012). Thus, BBB is thought as a central target for abetting neuroprotection and neurorestoration in stroke. The BBB is mainly composed of four cellular elements: endothelial cells, astrocyte end-feet, microglial cells, and pericytes. A breakdown in any of its individual components may contribute to BBB dysfunction (Sandoval and Witt 2008). BBB impairment is a well-known marker of secondary cerebral injury following acute stroke associated with an increase in vascular permeability and brain edema, exacerbating the initial ischemic injury (Latour et al. 2004). The breakdown of the BBB during ischemic stroke constitutes the main cause of early death after a stroke, but the molecular mechanisms involved in these processes remain speculative. Aside from its function in water homeostasis, recent studies showed possible interrelations between Aquaporin-4 (AQP4) and BBB dysfunction followed by cerebral edema (Saadoun and Papadopoulos 2010). AQP4 is a water-channel protein expressed strongly in the mammalian brain, abundantly in astrocyte foot processes at the borders between the brain parenchyma and major uid compartments, including cerebrospinal uid and blood (Papadopoulos and Verkman 2007). The role of AQP4 has been explored in ischemic stroke where it participates in the formation of cerebral edema (Ribeiro Mde et al. 2006). Simultaneously, AQP4 facilitated the clearance of vasogenic cerebral edema in pathologies where edema uid accumulates in the extracellular space Animals and grouping Adult male Sprague Dawley rats weighing 230280 g were provided by Shanghai Laboratory Animal Center (NO., SCXK, Shanghai, 2007-0005). Animals were housed in a room with temperature of 2123 C, relative humidity of 3070%, and a 12-h light/12h dark cycle (lights on at 08:00 h). They had free access to food and water. All animal experiments was conducted in accordance with the Guide for the Care and Use of Laboratory Animals issued by National Academy of Sciences, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. All procedures used in this study were approved by the local ethical committee for animal research. A total of 300 rats were randomly divided into 5 groups (n = 60 per group). They are sham-operated group, middle cerebral artery occlusion (MCAO) model group, Rg1-treated group, acetazolamidetreated group, and Rg1 plus acetazolamide-treated group. Of the 5 groups, each group was further divided into 5 sub-groups according to the time points of 6 h, 1 d, 3 d, 7 d and 14 d with 12 rats per sub-group after ischemia/reperfusion. Rg1 was dissolved in normal saline to the concentration of 5 mg ml1 , and acetazolamide was dissolved in soybean oil to the concentration of 25 mg ml1 . Rats in Rg1-treated group, acetazolamide-treated group, and Rg1 plus acetazolamide-treated group were given intraperitoneal injections of Rg1 (20 mg kg1 ) plus soybean oil (100 mg kg1 ), acetazolamide (100 mg kg1 ) plus normal saline (20 mg kg1 ) and Rg1 (20 mg kg1 ) plus acetazolamide (100 mg kg1 ), respectively. Rats in sham-operated group and MCAO model group just received normal saline and soybean oil of same volume, respectively. Intraperitoneal injections were conducted twice per day and started 3 days before MCAO model establishment until the animals were killed. In each sub-group, 4 rats were used to assess AQP4 expression in striatum of lesion side by immunohistochemical analysis and real-time uorescence quantitative polymerase chain reaction (PCR) respectively, and 4 rats were used to assess BBB permeability by measuring extravasated Evans blue (EB) dye in the brain tissue.

Focal cerebral ischemia/reperfusion Focal cerebral ischemia was induced by MCAO as described previously by Longa et al. (1989). Briey, rats were initially anesthetized by intraperitoneal injections of 10% chloral hydrate at a dose of 350 mg kg1 after 12 h fasting. A nylon lament was advanced from the external carotid artery into the lumen of internal carotid artery until the rounded tip reached the entrance to the middle cerebral artery. Following 120 min of MCAO, rats were re-anaesthetized and the occluding lament was withdrawn gently in order to allow reperfusion to take place. Sham-operated rats underwent the same surgical procedure, except that the lament was not inserted. Body temperature was maintained at 3738 C using a heating pad. The experimenter was blinded to the treatment that the rats had received prior to all subsequent analyses.

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Neurological decit scores Neurological examinations were performed at 6 h, 1 d, 3 d, 7 d and 14 d after ischemia/reperfusion by a blinded observer using a 5-point scale published previously by Longa et al. (1989). Rats were assessed a score between 0 and 4: 0, normal motor function; 1, exion of the contralateral forepaw upon lifting of the animal by the tail; 2, circling to the contralateral side; 3, falling to the contralateral side; and 4, no spontaneous walking with a depressed level of consciousness. Only rats with neurological scores of 13 were considered successful models and used in our study. Measurement of BBB permeability EB dye was used to investigate the effects of Rg1 on BBB permeability. BBB permeability was assessed by measuring extravasated EB dye in the brain tissue. EB dye saline solution (4 ml/kg, 2%) was administered intravenously via the femoral vein and allowed to circulate for 90 min before the rats sacriced. The animals were then perfused with normal saline through the left ventricle after which, brains were removed and dissected. Each hemisphere was weighed, then heated in water bath for 24 h after the addition of 3 ml of methanamide. Samples were then centrifuged for 20 min at 5000 rpm and 10 min at 10,000 rpm. The absorbance of the supernatants was measured at 632 nm with a spectrophotometer (UV-7502PC, Shanghai Xinmao Instrument Co., Ltd., China) in order to assess the leakage. Tissue processing and immunohistochemical analysis Immunohistochemical staining was used to evaluate whether treatment with Rg1 changes the expression of AQP4 after cerebral ischemia/reperfusion. Four coronal sections (4 m thick) at the level of the anterior commissure in the infarct region were obtained. For IHC analyses, the brain slices were postxed in 4% paraformaldehyde for 24 h and then gradient alcoholic dehydrated and embedded in parafn. After that, sections were deparafnized, antigen repaired for 3 min in sodium citrate buffer (pH 6.0) and incubation with 0.3% H2 O2 in PBS. Sections were then incubated with 1:200 mouse anti rat AQP4 monoclonal antibody overnight at 4 C. After washing in PBS, brain sections were incubated with the primary antibodies of IHC detection kit (PV-9005, ZSGB-BIO Co., Peking, China) for 20 min at 37 C and with the secondary antibodies at 37 C for 30 min, then visualized using 3,3 -diaminobenzidine tetrahydrochloride (DAB kit, ZLI-9018, ZSGB-BIO Co., Peking, China). Last, sections were counter stained with hematoxylin and then gradient alcoholic dehydrated. Sections were photographed from 5 different angles and slides were analyzed by professional image analysis software (Image-Pro Plus 6.0) for semi-quantitative analysis of AQP4 expression according to the integrated optical density (OD) and gray value of positive cells. All procedures were carried out in a double-blinded manner. Real-time uorescence quantitative PCR Total RNA for AQP4 mRNA determination was isolated from ipsilateral striatum and then reverse transcribed. The reverse transcription reaction was performed in a nal volume of 20.55 l containing 2 l of RNA template, 5 l of random primer N6 (100 M), 10 l of 2 reverse transcription buffer, 3 l of dNTP (2.5 mM), 0.4 l of MMLV Rtase (200 U/l), and 0.15 l of Rnasin (40 U/l). After incubating at 37 C for 60 min, the reverse transcription reaction was terminated by heating at 85 C for l0 min and then hold at 4 C. Obtained cDNA was then amplied by PCR using the following primers: for AQP4, forward 5 -CAGAACCAAGGCGTAGACCG-3 , reverse 5 -TCCCTGGAAATGACTGAGAAA-3 , product size: 257 bp;

for DAPDH, forward 5 -GTGCTGAGTATGTCGTGGAGTCT-3 , reverse 5 -GGAAGGGGCGGAGATGA-3 , product size: 104 bp. The totally 20 l reaction mixture of PCR consisted of 2 l of cDNA template, 10 l of 2 Real-time PCR Master Mix, 0.4 l Tap DNA polymerase (2.5 U/l), 0.08 l mRNA F primer (20 M), 0.08 l mRNA R primer (20 M), and 7.44 l double distilled water. Statistical analysis Statistical analyses were performed using SPSS 13.0. All experimental data were expressed as mean standard deviation. Neurological score data were analyzed using a repeated measures analysis-of-variance (ANOVA). When sphericity could not be assumed (Mauchlys sphericity test: p < 0.05), p-values were adjusted using the GreenhouseGeisser correction. Paired t-test was used for the signicant difference of EB content between left and right hemisphere. Comparison between multiple groups were done using one-way ANOVA and comparison between two groups were done using two sample t-test. p values of less than 0.05 were considered statistically signicant. Results Rg1 improved neurological function Sham-operated rats did not show visible neurological decits. Neurological scores (corresponding to the severity of neurological decits) peaked at 3 d and descended gradually for 14 d in the MCAO model group. After Rg1-treated, acetazolamide-treated or Rg1 plus acetazolamide-treated, neurological decit scores could continuously reduce at 6 h, 1 d, 3 d, 7 d and 14 d after ischemia/reperfusion (Fig. 2A). General linear model with repeated measures analysis of variance for neurological scores at 5 repeated measures showed that Rg1-treated group (1.00 0.08), acetazolamide-treated group (1.03 0.08) and Rg1 plus acetazolamide group (0.70 0.08) could signicantly reduce the changing trend of neurological decit scores when Compared with the MCAO model group (1.55 0.08) (p < 0.05), and Rg1 plus acetazolamide treatment could further reduce the changing trend of neurological decit scores when compared with the Rg1 and acetazolamide treatment, respectively (p < 0.05) (Fig. 2B). Rg1 reduced BBB disruption BBB disruption occurred at 6 h after ischemia/reperfusion and reached to the maximum at 3 d, then started to decline. The nonischemic hemispheres were not signicantly different between groups (p > 0.05). Compared with sham-operation group, the EB content of model group signicantly increased at each time point (p < 0.05). Compared with model group, ginsenoside Rg1 group, acetazolamide treatment, and ginsenoside Rg1 plus acetazolamide group all have signicantly decreased EB content at each time point (p < 0.05). Compared ginsenoside Rg1 group with ginsenoside Rg1 plus acetazolamide group, there were statistically signicant differences of EB content at the time points 6 h, 1 d, 3 d after ischemia/reperfusion (p < 0.05), whereas there were not at the time points of 7 d and 14 d (p > 0.05) (Fig. 3). Rg1 downregulated the expression of AQP4 Under the microscope, the positive cells were brown and AQP4 was found expressing on the cell membrane but not in the nucleus and cytoplasm. Cavitation and rarefaction of cytoplasm were shown in the swelling cells. The results of immunohistochemical analysis of AQP4 protein expression (Figs. 4 and 5) and real-time uorescence quantitative PCR of AQP4 mRNA expression (Fig. 6)

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Fig. 2. (A) Effects of ginsenoside Rg1 on neurological decits after ischemia/reperfusion in rats ( x s, n = 12). (B) General linear model with repeated measures analysis of variance for the changing trend of neurological scores. *p < 0.05 compared with middle cerebral artery occlusion (MCAO) group. p < 0.05 compared with Rg1.

MCAO model group at each time point (p < 0.05) (Figs. 46). Compared with model group, Rg1 group, acetazolamide treatment, and Rg1 plus acetazolamide group all have signicantly decreased AQP4 expression at each time point (p < 0.05) (Figs. 46). Compared Rg1 group with Rg1 plus acetazolamide group, there were statistically signicant differences of AQP4 expression at the time points 6 h, 1 d, 3 d after ischemia/reperfusion (p < 0.05), whereas there were not at the time points of 7 d and 14 d (p > 0.05) (Figs. 46). Discussion The present study in rats provided the rst evidence that Rg1 provides neuroprotection against BBB disruption and neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of AQP4. The main ndings of present study were that Rg1 could improve neurological injury, attenuate BBB disruption and downregulate AQP4 expression induced by cerebral ischemia/reperfusion insults in rats. These results suggested that Rg1 as a neuroprotective agent may be potentially used for acute ischemic stroke. Preclinical models of stroke are essential for understanding the basic mechanisms of ischemic damage and functional recovery

Fig. 3. Effects of ginsenoside Rg1 on Evans blue (EB) leakage after ischemia/reperfusion in rats ( x s, n = 4). # p < 0.05, compared with the shamoperation group; *p < 0.05 compared with middle cerebral artery occlusion (MCAO) group. p < 0.05 compared with Rg1.

both showed that AQP4 expression was increased begun with 6 h after ischemia/reperfusion, then reached to the maximum at 3 d and still remained at a high level at 7 d. Compared with the shamoperation group, the AQP4 expression increased signicantly in the

Fig. 4. Immunohistochemistry analysis of aquaporin-4 (AQP4) expression in the striatum at 3-day time point after ischemia/reperfusion (n = 4). Sham: Sham-operated group; model: middle cerebral artery occlusion model group; Rg1: Rg1-treated group; AZA: acetazolamide-treated group; Rg1AZA: Rg1 plus acetazolamide-treated group (immunohistochemistry, 400).

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Fig. 5. Semi-quantitative analysis of AQP4 expression according to the integrated density (OD) and gray value of positive cells by a professional image analysis softx s, n = 4). # p < 0.05, compared with the sham-operation ware Image-Pro Plus 6.0 ( group; *p < 0.05 compared with middle cerebral artery occlusion (MCAO) group. p < 0.05 compared with Rg1.

thereafter as well as for the initial testing of neuroprotective compounds. Rat models for stroke are and will long be the rst choice because they are similar vasculature to humans, low cost, a number of well described behavioral outcome measures compared with those in mice (Lipsanen and Jolkkonen 2011). Transient MCAO using the intraluminal thread is the most common used reperfusion model in rats (Longa et al. 1989), and the same authors developed a 04 neurologic grading scale to assess neurological function impairment following cerebral ischemia (Longa et al. 1989). In the present study, we applied this 5-point scale to evaluate extent of the neurological decit. The score in the model group was increased at 6 h of reperfusion, peaked at 3 d, then declined with time going. In Rg1-treated group, the score was decreased from the beginning and neurological decits were signicantly attenuated compared with the model group at each time point. These results strongly suggest Rg1 treatment promotes neurological functional recovery after stroke. When combined Rg1 with acetazolamide, which is a reversible inhibitor of AQP4 (Tanimura et al. 2009), the functional recovery was stronger and more stable. Disruption of BBB is a critical event during cerebral ischemia, followed by passive diffusion of water leading to vasogenic edema and secondary brain injury. Cerebral edema is a major and potentially fatal complication of acute ischemic stroke. Therefore, BBB has been one of major targets for the understanding of mechanisms mediating cerebral ischemia damage and for development of new treatment for ischemic stroke (ODonnell et al. 2004). In the present study, we studied the effect of Rg1 on BBB disruption and the data showed that Evans blue content, as an indicator of BBB

Fig. 6. Real-time uorescence quantitative PCR analysis of AQP4 mRNA expression x s, n = 4). # p < 0.05, compared with the in the striatum after ischemia/reperfusion ( sham-operation group; *p < 0.05 compared with middle cerebral artery occlusion (MCAO) group. p < 0.05 compared with Rg1.

permeability, distinctly increased in the ipsilateral hemisphere at each time point after ischemia/reperfusion and Rg1 treatment signicantly attenuated the leakage of EB. These data showed that Rg1 could reduce BBB disruption to some extent after cerebral ischemia/reperfusion. The neuroprotective effect of Rg1 has well been established in the many other studies in the recent decades, and their mechanisms revealed that Rg1 can increase hypoxia inducible factor-1 levels, activate nitric oxide synthase activity and N-methyl-d-aspartate receptors as well as glucocorticoid receptors and L-type voltagedependent Ca2+ channels, and upregulated vascular endothelial growth factor expression (Jiang et al. 2012; Shen and Zhang 2007; Tang et al. 2011; Zhang et al. 2008). In the present study, we studied the effect of Rg1 on expression levels of AQP4 at the same time point as for Evans blue leakage. Consistent with the result of EB, the data showed that AQP4 was upregulated and markedly expressed in the ipsilateral hemisphere at each time point after ischemia/reperfusion and Rg1 treatment signicantly inhibited the expression of AQP4. These results supported that AQP4 is involved in the BBB breakdown after cerebral ischemia and reperfusion. Thus, Rg1 treatment reduced AQP4 expression corresponded to its protective effect against BBB breakdown, further suggesting that Rg1 protects BBB integrity after ischemia/reperfusion by blocking AQP4. It is worth noting that the neuroprotective activity of Rg1 for acute ischemic stroke may raise a concern that all Ginsenosides possess this activity because several other Ginsenosides, such as Rh2 (Park et al. 2004), Rb and Ro (Zhang et al. 2006), Rg3 (Tian et al. 2009), and Rd (Ye et al. 2011), also have neuroprotective effects in ischemic brain injury. However, we cannot conclude that all ginsenosides possess neuroprotective activity due to the following reasons: (1) Ginsenosides have been divided into two groups: PPDtype and PPT-type. In the PPD group, sugar residues are attached to the -OH at C-3 and/or C-20. In the PPT group, sugar moieties are attached to the -OH at C-6 and/or -OH at C-20 (Qu et al. 2009). Since there are big differences in their chemical structures, it is hardly to conclude that all Ginsenosides have same biological activity from the point view of chemistry. (2) The ginsenosides potent anti-oxidative activity plays an important role on the neuroprotecive effects in insulted ischemic brains (Siddique et al. 2000). However, ginsenoside aglycones have different antioxidant abilities. Ginsenosides Rg2, Rg3 and Rh2 were prooxidative; ginsenosides Rb1, Rc, R1, Rd, Re, Rb3, Rg1, and Rh1 functioned as antioxidants (Liu et al. 2003). In an in vitro cortical cell model, only ginsenoside Rb1 and Rg3 exerted efcacious in protecting neurons from oxidative damage, whereas other ginsenosides such as Rb2, Rc, Re, Rg2, Rh1, and Rh2, Rg1 and Rg5 had no neuroprotective activities (Kim et al. 1998). It suggested that the neuroprotective activities of different ginsenosides may be various. (3) Although several ginsenosides showed neuroprotective activity, most of the studies in this eld are explanatory on the therapeutic potential of ginseng compounds with little explanation of mechanism of action, especially on the causal relationship of the molecular or biological changes induced by ginsenosides on therapeutic action. Even if the similar neuroprotective activity was found in two ginsenoside groups such as Rg3 (Tian et al. 2009) and Rg1 in the present study, the anti-ischemic effects of Ginsenosides may have different molecular and biological mechanism of actions. Tian et al. (2009) indicated that PPD-type ginsenoside Rg3 could inhibit the opening of mitochondrial permeability transition pores by free radical scavenging action in the rat brain, suggesting its mechanism of neuroprotective actions as an antioxidant and inhibitor of the mitochondrial permeability transition pores. In the present study, we indicated that a novel molecular mechanism, regulation of AQP4 expression by PPT-type ginsenosides Rg1, is involved in the potent neuroprotective actions, and this effect of AQP4 on the

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neuroprotective activity of Rg1 has not yet been reported. Thus, whether the neuroprotective effects of different Ginsenosides in acute ischemic stroke may have same or different molecular and biological mechanisms is worthy of further exploration. In conclusions, the present study demonstrated that Rg1 can improve neurological function decits and reduce BBB disruption after focal cerebral ischemia. The mechanism of this neuroprotection by Rg1 in cerebral ischemia/reperfusion may be through downregulation of AQP4 expression. Conict of interest No conict to disclose. Acknowledgments This project was supported by the grant of National Natural Science Foundation of China (81173395/H2902), the Key Project of Wenzhou Municipal Science and Technology Bureau of Zhejiang Province, China (Y20070038). References
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Please cite this article in press as: Zhou, Y., et al., Ginsenoside Rg1 provides neuroprotection against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of aquaporin 4 expression. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2013.12.005